Cells committed to formation of the eye arise within the anterior neural plate during early development of the brain. It is our long term coal to understand the molecular basis of their origin and later development into ocular tissues. Such understanding would be of relevance to the- diagnosis and possible prevention of many defects in eye development that are of genetic or environmental origin. In addition, this knowledge could ultimately lead to methods for the in vitro culture of human retinal stem cells, and the use of such stem cells in efforts to restore vision by cell transplantation. The scope of this proposal is to identify inductive interactions that regulate cell fate during the establishment of the eye. We will carry out surgical manipulations on the early chick embryo to infer the presence of such interactions between tissues. The use of wholemount in situ hybridization to detect specific messenger RNAs will allow us to directly visualize the changes in gene expression that precede and accompany the formation of ocular structures in the embryo. One such molecular marker, the Pax-6 gene, is a highly conserved regulator of transcription that is essential to eye development. It is associated with aniridia, a human genetic disorder affecting formation of the eye. In the early embryo, Pax-6 is expressed at high levels in the optic vesicle and early lens placode, as well as the neural and ectodermal primordia which give rise to these structures. Another marker, which we have recently isolated and designated lOA6, is a gene expressed almost exclusively within the early optic cup and retina. Specifically, we will first determine whether certain regions of mesoderm in the chick gastrula are required for the establishment of a single, cyclopic eye forming region. We will then determine whether mesoderm underlying the emerging neural plate shapes this eye forming region into two separate primordia. The establishment of clear assays for such interactions provides a basis for future experiments designed to approach their molecular nature. At another crucial stage, the early forebrain has developed two outgrowths known as optic vesicles. We will determine whether the overlying head ectoderm, which is fated to become the lens, provides signals essential to the growth and formation of these vesicles into optic cups. The optic cup represents a key step in the spatial organization and establishment of the retina, iris, ciliary body and pigment epithelium. Finally, we will investigate the possibility that cell cultures undergoing lens differentiation can provide a source of inductive factors which act upon the optic vesicle. This would make possible the biochemical study and eventual identification of such signalling molecules.